Nucleic Acids Research 2005 33(Database Issue):D403-D406; doi:10.1093/nar/gki099
Nucleic Acids Research, 2005, Vol. 33, Database issue D403-D406
© 2005, the authors
Nucleic Acids Research, Vol. 33, Database issue © Oxford University Press 2005; all rights reserved
SilkSatDb: a microsatellite database of the silkworm, Bombyx mori
M. D. Prasad,
M. Muthulakshmi,
K. P. Arunkumar,
M. Madhu,
V. B. Sreenu1,
V. Pavithra1,
B. Bose1,
H. A. Nagarajaram1,
K. Mita2,
T. Shimada3 and
J. Nagaraju*
Laboratories of Molecular Genetics and 1 Computational Biology and Bioinformatics Facility (EMBnet India Node), Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad 500076, India, 2 Insect Genome, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan and 3 Laboratory of Insect Genetics and Bioscience, Department of Agricultural and Environmental Biology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
* To whom correspondence should be addressed. Tel: +91 40 27171427; Fax: +91 40 27155610; Email: jnagaraju{at}cdfd.org.in
Received May 27, 2004; Revised and Accepted October 14, 2004
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ABSTRACT
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The SilkSatDb (
silkmoth micro
satellite
data
base) (
http://www.cdfd.org.in/silksatdb)
is a relational database of microsatellites extracted from the
available expressed sequence tags and whole genome shotgun sequences
of the silkmoth,
Bombyx mori. The database has been rendered
with a simple and robust web-based search facility, developed
using PHP. The SilkSatDb also stores information on primers
developed and validated in the laboratory. Users can retrieve
information on the microsatellite and the protocols used, along
with informative figures and polymorphism status of those microsatellites.
In addition, the interface is coupled with Autoprimer, a primer-designing
program, using which users can design primers for the loci of
interest.
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INTRODUCTION
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Microsatellites are short tandem repeats of 1–6 bp, which
are ubiquitous in both prokaryotes and eukaryotes both in protein-coding
and non-coding regions (
1). Microsatellites are used widely
in a variety of applications including genetic distance measures
and phylogeny reconstruction, population genetics, genetic mapping,
elucidating evolutionary history, artificial selection and forensics
(
2,
3). As the whole genome sequence data of many organisms are
now available, an unambiguous picture of occurrence and genomic
distribution of microsatellites is emerging, and the study of
microsatellites has attained importance to address various biological
questions.
The silkworm, Bombyx mori is an important economic insect and lepidopteran molecular model (4). Study of microsatellites in this insect will help in genetic fingerprinting of diverse silkmoths, construction of molecular linkage map, marker assisted selection, in addition to the basic understanding of microsatellites (5,6). Evolutionarily conserved microsatellite loci of B.mori can further be extended to study other lepidopteran insects, which include some of the most destructive agricultural pests. With expressed sequence tag (EST) sequences published (7) and sequenced whole genome shotgun (WGS) sequences prepared for silkworm (8), an effort has been put forth to create a database of microsatellites called SilkSatDb. The data generated will be useful for the research community working on insect genetics, particularly lepidopteran geneticists across the globe.
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STRUCTURE OF THE DATABASE
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The SilkSatDb is an online relational database that catalogues
information about the microsatellite repeats of the silkworm.
The design of the database schema basically follows the ‘Three-level
schema architecture’ as shown in
Figure 1. The database
stores three kinds of data: the microsatellite repeats found
in
B.mori EST and WGS sequences, sequence details and the primers
developed for these microsatellites. Currently, the database
encompasses information pertaining to 12 000 WGS contigs downloaded
from DDBJ (
http://www.ddbj.nig.ac.jp/anoftp-e.html), 9300 EST
and 50 genomic sequences of
B.mori. Each entry in the sequence
information table includes an identifier for the sequence type
and the accession number to which it belongs. The accession
number is a number, possibly with a few characters in front,
that uniquely identifies the sequence stored in the flat-file
format. Since ESTs are a large collection, information is stored
with respect to their cDNA library clone names (based on the
annotation from
http://www.ab.a.u-tokyo.ac.jp/silkbase/).
The extraction of microsatellite repeats is done using Simple
Sequence Repeat Finder (SSRF) program (
9,
10), which is written
in C. This program scans a given DNA sequence and identifies
non-redundant, perfect microsatellite tracts. Repeats information
extracted is categorized (except EST) as mono, di, tri, tetra,
penta and hexa repeats and placed in their respective sequence
groupings. The EST sequence repeats are classified according
to their library type.
In addition to the sequence information as mentioned above the database also includes a list of primers, designed and tested in our laboratory, for about 200 loci, with their respective PCR amplification conditions. Figure 2 illustrates the entire relational schema of SilkSatDb.
Besides storing data on microsatellites, SilkSatDb hosts other
useful details of microsatellites such as their frequencies,
different types of mutations prevalent in them, their allelic
frequency, mapping populations and their evolutionary conservation
in heterologous silkmoths (
11). The protocols for microsatellite
analysis and the recently improvised methodology for the inter
simple sequence repeats (ISSRs)-based genotyping are also listed
with experimental details. Thus, the whole website forms an
integrated site wherein the
in silico analysis is extended to
bench, providing the user everything about silkmoth microsatellites
and microsatellite-based markers. To the best of our knowledge,
this is the first site on microsatellites and microsatellite-based
genetic markers in insects outside
Drosophila.
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DATA EXTRACTION
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The user-friendly interface for the database has been developed
using PHP, a server side scripting language. This acts as a
comprehensive and integrated resource for retrieval of the information
from SilkSatDb. The user can query for microsatellites, extracted
from WGS and EST sequences, using the repeat type/motif and
the number of occurrences of the repeat.
The query results are displayed in a tabular format showing the accession number, the motif, its frequency and location in the sequence. Hyper links are provided for the motif and the accession number in the table. The motif link takes the user to the detailed description of the motif occurrence in each sequence. The accession number link connects to its respective sequence information. Selection buttons are provided for each of these accession numbers in the table, by which the user can get the available primers corresponding to the microsatellite tracts. If the primers are not available, the interface links to ‘Autoprimer’ (9,10), an automated primer design software that takes the sequence data along with flanking regions and other primer parameters such as primer length, GC content, Tm etc., as an input for the designing of the primers (Figure 3).
View larger version (83K):
[in this window]
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Figure 3. Symbol display of query form for repeat motif search, output format for microsatellite loci and the primer designing using ‘Autoprimer’ primer-designing program for the selected locus.
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FUTURE PERSPECTIVES
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Plans for future database releases include incorporation of
additional microsatellite loci to the existing data as and when
they are developed and validated. The construction of the microsatellite
linkage map of silkworm is underway and the mapping data will
be hyperlinked to the SilksatDb when it becomes available. The
EST sequencing projects from various lepidopterans are underway
in several laboratories. In future, the database will be updated
with microsatellite loci extracted from the genome and EST sequences.
Also, the microsatellite loci of silkworm are being tested in
heterologous species, for their evolutionary conservation. These
data too will be uploaded when they become available, so that
the community of investigators from insect molecular biology
and molecular ecology can utilize this resource to address various
issues.
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ACCESS
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This is a new molecular genetic resource freely accessible for
research purposes for non-profit and academic organizations
at
http://www.cdfd.org.in/silksatdb. Comments, suggestions and
questions are welcome and should be directed to
jnagaraju{at}cdfd.org.in.
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ACKNOWLEDGEMENTS
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The authors acknowledge the help of Mrs. Geeta Thanu during
the database creation. This work was supported by a grant from
Department of Biotechnology, Government of India, New Delhi,
to J.N. H.A.N. gratefully acknowledges the core grant support
of CDFD. K.P.A. and V.B.S. are recipients of CSIR research fellowship.
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Notes
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The online version of this article has been published under
an open access model. Users are entitled to use, reproduce,
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is properly and fully attributed; the Journal and Oxford University
Press are attributed as the original place of publication with
the correct citation details given; if an article is subsequently
reproduced or disseminated not in its entirety but only in part
or as a derivative work this must be clearly indicated. For
commercial re-use permissions, please contact
journals.permissions{at}oupjournals.org.
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